Methylmalonyl-CoA mutase, the only member of the family that is found in both bacteria and in man, catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. Its dysfunction leads to methylinalonic aciduria, an inborn error of metabolism that, in severe cases, can be fatal. A few years ago, we bad reported a novel and anomalously large kinetic isotope effect on the cleavage of the cobalt carbon bond of the cofactor when an isotopic substitution from protium to deuterium was made in the substrate. This was interpreted as evidence that homolysis of the cobalt-carbon bond is coupled to hydrogen atom abstraction from the substrate leading to a substrate radical. The large deuterium isotope effect (35.6 at 20oC) suggests the contribution of tunneling to this hydrogen atom transfer although other explanations are also possible. This has recently been examined by monitoring the temperature dependence of the isotope effect which yields values for the ratio of the Arrhenius preexponential factors (AH/AD) and for the difference in activation energies (EaD-EaH) that lie well outside the semiclassical range. In order to draw mechanistic conclusions from the measured values of isotope effects it is necessary to estimate theoretically their values for alternative pathways. This proposal focuses on combining the experimental approach for evaluating tunneling that is a component of the parent grant, with theoretical calculations of kinetic isotope effects in the methylmalonyl-CoA mutase-catalyzed reaction under presteady-state conditions. We will use available crystallographic information to build a model of the active site and optimize structures of the reactants, transition states, and putative intermediates using semiempirical, DTF, and/or ab initio methods within recently developed QM/MM techniques. Vibrational analysis performed on these structures will allow us to calculate isotope effects within the semiclassical approximation. The tunneling contribution will be then calculated. These studies will allow us to better understand and control the mechanism of this novel reaction in the clinically important enzyme.